1. Field of the Invention
This invention relates to the immobilization of catalytically active proteins and more particularly to the immobilization of enzymes on inorganic support materials. Enzymes usually have high molecular weights, can catalyze numerous specific chemical reactions, and many techniques have been developed to physically or chemically immobilize enzymes on various support materials. The techniques were developed because enzymes are generally water soluble and thus are difficult to remove from the reaction media for reuse. The techniques were developed to immobilize or fix the enzymes on substantially water insoluble supports without loss of catalytic activity so that the enzyme, on its support, which is called the enzyme-support composite, could be used repeatedly. However, many currently used immobilization methods are not readily accomplished or reproduced, and tend to be expensive to use in commercial operations. The method of the present invention produces a reproducible composite with relative ease at a very low cost.
2. Description of the Prior Art
It is known that enzymes can be fixed or immobilized on both organic and inorganic supports. Enzymes can be immobilized on particles of various metal oxides so that the enzyme is bound or deposited on the surface of the oxide and is supported thereon to catalyze a specific reaction as shown for example in U.S. Pat. No. 3,850,751. This type of immobilization, while satisfactory, has the disadvantage that the enzyme tends to desorb or release itself from the surface of the support over a period of time. The activity of the composite gradually diminishes over the long term, and the sample being treated is contaminated with active enzyme which continues to react with the sample after it has been removed from the reaction chamber for analysis. Accordingly, data for a given sample or batch may be obtained which may not be truly representative of the nature of the sample.
Many techniques have been attempted to lessen the rate at which enzymes will desorb from a support structure. In U.S. Pat. No. 3,873,426, an enzyme and an organic dye, typically a halotriazine dye, are absorbed onto alumina below pH 7. This codeposition is stated to increase the affinity of the enzyme for the support. However, in such a technique, the enzyme and the added species in this example, the dye, can eventually desorb into the test sample at some finite rate. Therefore, another possible contaminent may be introduced into the test sample which may give false results when the sample being tested is examined spectrophotometrically or electrochemically.
U.S. Pat. No. 3,982,997 discloses the use of mixed metal oxides to immobilize enzymes and to increase the amount of deposition of the enzyme and stability of the composite. While with this technique the enzyme does exhibit an increased initial activity, it does not bind the enzyme to the support with an affinity which is a desired strength of binding.
Techniques also have been utilized to covalently link an enzyme to the support to circumvent the desorption problems encountered in the techniques discussed above. These techniques of covalent bonding have generally used a coupling agent which bonds to the support with one reactive group and bonds to the enzyme with another reactive group. These groups have generally been reactive species such as diazos, silanes, and the like. For examples of these coupling agents, reference is made to U.S. Pat. Nos. 3,930,951, and 3,519,538.
In other techniques, the enzyme is used as the acidic member in forming a salt pair with an anion exchange resin, e.g. U.S. Pat. No. 3,126,324. Similarly, many techniques of gel entrapment are known wherein the enzyme is trapped in a hydrogel while maintaining its catalytic activity, e.g. U.S. Pat. No. 3,223,593.
Some techniques involve cross-linking the deposited enzymes to each other. These methods involve the use of reactive cross-linking reagents such as glutaraldehyde, but the reagents may adversely affect the activity of the enzyme. Such a cross-linking procedure is disclosed in U.S. Pat. No. 4,004,979. Also, methods of using sulfhydryl groups on the enzyme to cross-link the enzymes to each other are known, e.g. U.S. Pat. No. 4,008,126. This sulfhydryl cross-linking, as disclosed in the above patent, requires that all proteins which are being deposited have a relatively high proportion of cysteine or cystine amino acid residues in their structure.
Although the method is satisfactory for enzymes having the relatively high proportion of cysteine or cystine residues, the method is not applicable to proteins having relatively low proportions of the residues.
Many of the above immobilization techniques have been used in large scale commercial operations, but have not been completely satisfactory, due to the expense in production of the composite, the lack of long-term stability of the composite, certain difficulties involved e.g. in purifying the coupling agents from the composite after its production, and in the gradual desorption of these reagents into delicate samples, or into electrode systems used to monitor product levels in reactors.